Quantitative and qualitative differences in enzymes synthesizing cell wall arabinose-containing polysaccharides will be compared in extracts from ethambutol (EMB)-sensitive and -resistant Mycobacterium smegmatis. Drug-sensitive enzyme(s) in cell socicates will be isolated, purified, characterized and used in cell-free systems. Purification techniques involve ion-exchange chromatography, gel chromatography, affinity chromattography and possibly by high pressure liquid chromatography (HPLC). The steps in enzyme purification will be monitored by gel electrophoresis and cell-free assays for oligosaccharide synthesis. Polysaccharides extracted from whole cells and from cell-free assays will be chemically hydrolyzed to monsaccharides, then derivatized to the corresponding alditol acetates which are identified by their retention times by HPLC when compared to standards. The long term goals are to characterize the drug-sensitive enzyme(s) and also explain the differences in enzymes extracted from related aerobic actinomycetes. Nocardia, Rhodococcus and Corynebacterium contain cell wall arabinogalactan (AG), but these genera are relatively resistant to EMB. The effect of EMB on mycobacterial cell walls will also be studied by immunoelectron microscopy. The binding of concanavalin A purified antibody to AG on thin sections of drug-sensitive and EMB-resistant bacteria will be measured with a colloidal gold label. We will test the hypothesis that CFZ does not enter drug-resistant isolates of M. smegmatis or other cells which are naturally CFZ- resistant. Incorporation of radioactive CFZ (if available) will be measured, or the transport of unlabelled CFZ through bacterial membranes. The competence in both academic and technical subjects acquired by students strengthens their preparation for and interest in rigorous biomedical research. Students will participate in enzyme extraction and purification, radioisotope use, electron microscopy and formal discussion of research involving drugs effective in treating tuberculosis and leprosy, diseases having a high incidence in minorities in this country.